1. Field of the Invention
The present invention concerns a method for the separation/removal of a mammalian serum albumin from a solution containing a mixture of proteins in order to obtain a solution/preparation that is substantially devoid of the serum albumin. The invention also concerns novel immobilized forms of albumin-binding ligands deriving from native forms of bacterial receptors that are able to bind to one or more serum albumins.
2. Technical Background
For a long time there has been a large demand for mammalian serum albumins, for instance serum albumin of human or bovine origin (HSA and BSA, respectively). For HSA this has mainly depended on its therapeutic use as a plasma volume expander. Originally serum albumins were obtained from sera/plasma of the appropriate species origin. For some years the focus has been to produce serum albumins recombinantly, in particular HSA. For bacterially produced recombinant forms, it has become urgent to remove host cell contaminants because they may be hazardous in vivo to mammals. For some time it has become apparent that producing HSA in transgenic animals should be beneficial, for instance in transgenic cows. This latter alternative, however, has the drawback that HSA will be present in mixture with the HSA analogue of the host animal (for instance with BSA if HSA produced in cows). This has created novel purification problems, for instance the specific removal of BSA from HSA.
Serum albumin preparations intended for use in vivo shall according to accepted practice contain less than 0,01%, such as less than 0,001% proteins that are heterologous to the species to which the preparation is to be administered. HSA preparations, for instance, that are to be used in humans shall contain less than 0,01%, such as less than 0,001% BSA. All percentages are w/w.
Removal of serum albumins has also been a concern when assaying samples containing serum albumin for other components. Blood derived samples such as whole blood, serum and plasma are typical examples. In this kind of samples serum albumin is one of most abundant substances and may disturb assays for other substances that are present in lower amounts. In analyzing protein components after, for instance, two-dimensional gel electrophoresis or mass spectrometric fragmentation, serum albumin will easily disturb.
Various forms of affinity chromatography and/or ion exchange chromatography have earlier been applied to the purification of serum albumins. For affinity chromatography the general goal has been to find a chromatographic media (ligand attached to a chromatographic base matrix) that provide the sufficient specificity in order to remove either predetermined contaminants or the serum albumin desired as the end product from complex mixtures.
3. Description of Related Art
Illustrative examples of ligands previously used and having selectivity for serum albumins are given by Theodore Peters in All about Albumin-Biochemistry, Genetics and Medical Applications, (Ed. Theodore Peters, Jr., Academic Press (1996) pages 77-126. There are also known other compounds that bind serum albumins, even with species selectivity, that for various reasons have not found use in the selective/specific removal and/or purification of serum albumins. Examples are albumin binding receptors (proteins) present on the cell surface of certain bacteria, typically streptococci. See for instance Nygren et al., Eur. J. Biochem. 193 (1990) 143-148 (Protein G), Guss et al., WO 9507300 (Protein MAG), Jonsson et al., Infect. Immun. 63 (1955) 2968-2975 (Protein ZAG). These bacterial receptors frequently also bind to other proteins, for instance Protein G to IgG and Protein MAG and ZAG to IgG and xcex12-macroglobulin. The various extra binding abilities of these proteins make them less suitable as ligands for the selective/specific removal of serum albumin from complex protein mixtures. The main use of their albumin-binding fragments has been as fusion partners, for instance in order to have an affinity handle attached to a protein to be purified. To the extent that immobilised forms have been produced it has been in order to make binding studies in relation to serum albumins from various mammalian species. See further Nygren et al (Eur. J. Biochem. 193 (1990) 143-148). Guss et al (WO 9507300) has outlined in a patent example to use intact protein MAG in an attempt to roughly purify albumin from mammalian blood. However it is apparent from Guss et al""s results that also IgG and xcex12-macroglobulin were bound together with albumin.
Additional examples of known bacterial receptors binding to serum albumin are Protein H and M proteins, both from streptococci.
Native forms of this kind of receptors typically have more than one subsequence that is responsible for binding to albumin or IgG. The receptors may contain 1, 2 or 3 albumin-binding and/or IgG-binding regions. Functionally similar regions may differ in sequence.
Objectives of the Invention
The first objective of the invention is to provide improved affinity methods for the removal of a serum albumin from a mixture of proteins in order to produce the serum albumin in pure form or a preparation essentially free of the removed serum albumin.
A second objective is to provide an affinity method as defined above which has an improved selectivity for a certain serum albumin that exists in mixture with one or more other serum albumins.
A third objective is to provide new affinity matrices carrying albumin-binding ligands having improved selectivities for serum albumins.
A fourth objective is an improved method for removal of serum albumin in samples that are to be assayed for one or more non-serum albumin components.
The methods for removal of serum albumin are based on matrices that carry an albumin binding ligand.
The Invention
It has now surprisingly been found that the albumin binding capacity of the type of bacterial cell surface receptors mentioned above may advantageously be utilized for the selective removal and/or purification of serum albumins.
The inventive method encompasses that a mixture which contains (a) a serum albumin that is derived for a certain mammalian species and (b) other compounds, in particular proteins, is contacted with a ligand under conditions permitting binding (adsorption) of the serum albumin to the ligand. The ligand is preselected to have affinity for the serum albumin and is attached to a base matrix or is possible to attach to a base matrix after having become bound to the serum albumin. The invention has the characterizing feature that the ligand derives from an albumin-binding form of a bacterial receptor having affinity for a serum albumin, said form not encompassing the IgG- or xcex12-macroglobulin-binding ability of native forms of these kind of receptors. The form is typically a fragment not encompassing the amino acid subsequences responsible for ability to bind to IgG- and/or xcex12-macroglobulin.